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Am J Physiol Heart Circ Physiol 296: H997–H1006, 2009.First published January 23, 2009; doi:10.1152/ajpheart.00660.2008.Muscle ring finger 1 mediates cardiac atrophy in vivoMonte S. Willis, 1,2 Mauricio Rojas, 1 Luge Li, 2 Craig H. Selzman, 6 Ru-Hang Tang, 3 William E. Stansfield, 3Jessica E. Rodriguez, 2 David J. Glass, 4 and Cam Patterson 1,51Carolina Cardiovascular Biology Center, 2 Department of Pathology and Laboratory Medicine, and 3 Department of Surgery,University of North Carolina, Chapel Hill, North Carolina; 4 Novartis Institutes for Biomedical Research Incorporated,Cambridge, Massachusetts; 5 Departments of Medicine, Pharmacology, and Cell and Developmental Biology,University of North Carolina, Chapel Hill, North Carolina; 6 Division of Cardiothoracic Surgery, University of Utah,Salt Lake City, UtahSubmitted 23 June 2008; accepted in final form 21 January 2009Willis MS, Rojas M, Li L, Selzman CH, Tang R, StansfieldWE, Rodriguez JE, Glass DJ, Patterson C. Muscle ring finger 1mediates cardiac atrophy in vivo. Am J Physiol Heart Circ Physiol296: H997–H1006, 2009. First published January 23, 2009;doi:10.1152/ajpheart.00660.2008.—Pathological cardiac hypertrophy,induced by various etiologies such as high blood pressure andaortic stenosis, develops in response to increased afterload and representsa common intermediary in the development of heart failure.Understandably then, the reversal of pathological cardiac hypertrophyis associated with a significant reduction in cardiovascular event riskand represents an important, yet underdeveloped, target of therapeuticresearch. Recently, we determined that muscle ring finger-1 (MuRF1),a muscle-specific protein, inhibits the development of experimentallyinduced pathological; cardiac hypertrophy. We now demonstrate thattherapeutic cardiac atrophy induced in patients after left ventricularassist device placement is associated with an increase in cardiacMuRF1 expression. This prompted us to investigate the role ofMuRF1 in two independent mouse models of cardiac atrophy:1) cardiac hypertrophy regression after reversal of transaortic constriction(TAC) reversal and 2) dexamethasone-induced atrophy. Usingechocardiographic, histological, and gene expression analyses, wefound that upon TAC release, cardiac mass and cardiomyocyte crosssectionalareas in MuRF1 / mice decreased 70% less than in wildtype mice in the 4 wk after release. This was in striking contrast towild-type mice, who returned to baseline cardiac mass and cardiomyocytesize within 4 days of TAC release. Despite these differences inatrophic remodeling, the transcriptional activation of cardiac hypertrophymeasured by -myosin heavy chain, smooth muscle actin, andbrain natriuretic peptide was attenuated similarly in both MuRF1 /and wild-type hearts after TAC release. In the second model,MuRF1 / mice also displayed resistance to dexamethasone-inducedcardiac atrophy, as determined by echocardiographic analysis. Thisstudy demonstrates, for the first time, that MuRF1 is essential forcardiac atrophy in vivo, both in the setting of therapeutic regression ofcardiac hypertrophy and dexamethasone-induced atrophy.ubiquitin ligase; cardiac hypertrophy; cardiac atrophy; left ventricularassist devicePATHOLOGICAL CARDIAC HYPERTROPHY develops in response toincreases in afterload and represents a common intermediary inthe development of heart failure, a leading cause of mortality inthe United States. Left ventricular (LV) hypertrophy is anindependent risk factor for several adverse outcomes, includingcardiac mortality, arrhythmias, and myocardial infarction (18,Address for reprint requests and other correspondence: M. S. Willis, Dept.of Pathology and Laboratory Medicine, Carolina Cardiovascular BiologyCenter, Univ. of North Carolina, Medical Biomolecular Research Bldg., Rm.2336, 103 Mason Farm Rd., Chapel Hill, NC 27599-7525 (e-mail: monte_willis@med.unc.edu).25, 28, 29). Results from numerous studies have suggested thatreducing heart mass in patients with pathological cardiac hypertrophymay reduce morbidity and mortality and improvespatient outcomes (6, 10, 26, 35, 36, 44, 45). However, the onlytreatments currently proven to reverse both structural andfunctional cardiac abnormalities associated with pathologicalcardiac hypertrophy are antihypertensive therapies and aorticvalve replacement (for aortic stenosis), both of which have alimited success rate (14, 39). Understanding the underlyingprocesses regulating the plasticity of the heart will allow us toidentify specific pathways against which to target new therapiesand may improve the long-term outcomes of patients withpathological cardiac hypertrophy.Muscle ring finger (MuRF) family proteins are striatedmuscle-specific proteins involved in cardiomyocyte developmentand the regulation of muscle mass (3, 32). MuRF1localizes specifically to the M line, a central structure of thesarcomere thick filament that has recently been recognized asa center of mechanical sensing (21). The ring finger domain ofMuRF1 has ubiquitin ligase capabilities (34), targeting sarcomericproteins such as troponin I and -/slow myosin heavychain (MHC) for degradation (13, 24). This degradation occursthrough the coordinated placement of polyubiquitin chains onrecognized substrates, which are subsequently degraded by theproteasome (50). MuRF1 also interacts with and inhibits serumresponse factor (SRF) activity, a transcription factor critical tothe development of cardiac hypertrophy (49). MuRF1’s localizationin the sarcomere places it in a unique position to bothrecognize the mechanical stresses that induce cardiac hypertrophyand regulate its subsequent development by controllingthe degradation of targeted sarcomeric proteins. Indeed, resultsfrom studies using animal models where the expression ofMuRF1 has been altered have implicated MuRF1 in the regulationof the development of cardiac hypertrophy: increasingMuRF1 in cardiomyocytes inhibits the development of hypertrophy(2), whereas the complete lack of MuRF1 results in thedevelopment of an exaggerated cardiac hypertrophy in responseto pressure overload (49).The present study was prompted by our observation thathuman cardiac tissue samples obtained from patients afterplacement of a LV assist device (LVAD) expressed significantlyhigher levels of MuRF1 protein than samples takenbefore the device was implanted. The unloading of the heart bya LVAD device leads to a decrease in the workload of theThe costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Downloaded from ajpheart.physiology.org on May 11, 2009http://www.ajpheart.org0363-6135/09 $8.00 Copyright © 2009 the American Physiological SocietyH997
H998MURF1 MEDIATES CARDIAC ATROPHY IN VIVOfailing heart and a decrease in LV mass. This form of cardiacatrophy is beneficial to the patient as it decreases the stress onthe heart. Although previous studies have identified thatMuRF1 is essential for the inhibition of the development ofpathological cardiac hypertrophy, there is no evidence linkingMuRF1 to the reversal of hypertrophy (i.e., cardiac atrophy). Inthis report, we used two independent models to demonstratethat MuRF1 is a necessary and significant mediator in theregulation of cardiac atrophy in vivo.MATERIALS AND METHODSAnimals. The MuRF1 / mice used in these experiments havepreviously been described (3, 49).Human cardiac LVAD samples. MuRF1 protein levels were determinedfrom heart samples from patients undergoing cardiac unloadingby the placement of a LVAD. Heart samples were collected frompatients with end-stage ischemic heart disease who received a LVADfor decompensated heart failure as a bridge to transplantation aspreviously described (40, 53). During the placement of the LVAD, acore of tissue is removed to prepare the LV for LVAD inflow. Thisspecimen was collected at the time of LVAD placement (pre-LVADsample), snap frozen in liquid nitrogen, and stored at 80°C. Whenpatients returned for a heart transplant, ventricular samples wereexcised just adjacent to the LVAD placement site (post-LVAD sample),snap frozen, and stored at 80°C. Use of the human tissue usedin this study was approved by the University of North CarolinaInstitution Review Board (no. 03-1359).Mouse cardiac hypertrophy reversal. Twelve to fifteen-week-oldMuRF1 / and wild-type (WT) mice were subjected to the reversible(slipknot) transaortic constriction (TAC) procedure recently describedby our laboratory (42). All experiments used 50% male and 50%female MuRF1 / mice and WT littermate controls. All animalprotocols were reviewed by the University of North Carolina InstitutionalAnimal Care Advisory Committee and were in compliance withthe rules governing animal use published by the National Institutes ofHealth.Dexamethasone model of cardiac atrophy. MuRF1 / and WTlittermates were maintained on a standard diet with unlimited access towater. Dexamethasone (5 mgkg 1 day 1 ) or saline vehicle was givenby a daily subcutaneous injection for 2 wk as previously described (1,16). In additional experiments, dexamethasone (1 mgkg 1 day 1 )orDMSO vehicle was continuously infused by a dorsally implanted osmoticminipump (model 2002, Alzet, Palo Alto, CA) for 2 wk. Echocardiographywas performed at baseline and after 2 wk of dexamethasonetreatment.Echocardiography. Echocardiography on mice was performed on aVisualSonics Vevo 660 ultrasound biomicroscopy system as previouslydescribed (49).Hemodynamic assessment of aorta flow. To assess the aorticconstriction after TAC and TAC reversal, right and left carotid arteryflow were assessed using a 20-MHz probe driven by a high-frequencypulsed Doppler signal processing workstation (Induc Instruments,Houston, TX) as previously described (42).Total RNA isolation/real-time PCR determination of mRNA expression.Total RNA was isolated from cardiac ventricular tissue aspreviously described (49). mRNA expression was determined using atwo-step reaction. cDNA was made using a High Capacity cDNAArchive kit (Applied Biosystems, Foster City, CA). PCR productswere amplified on an ABI Prism 7900HT Sequence Detection Systemusing cDNA and either 1) the TaqMan probe set in TaqMan UniversalPCR Master Mix or 2) unlabeled primers in Power CYBR GreenMaster Mix. The TaqMan probes used in these experiments includedbrain natriuretic peptide (BNP; Mm00435304_g1), smooth muscle-actin (Mm00808218_g1), -MHC (Mm00600555_m1), MuRF1(Mm01188690_m1), MuRF2 (Mm01292963_g1), atrogin-1/ muscleatrophy F box/F box only protein 32 (Mm00499518_m1), and 18S(Hs99999901_s1) (Applied Biosystems). The unlabeled primers formouse tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, matrixmetalloproteinase (MMP)-2, pro-collagen type I (ColI), procollagentype III (ColIII), laminin B (Lamb), and GAPDH were aspreviously published (43). Samples were run in triplicate, and relativemRNA expression was determined using 18S (TaqMan Probes) orGAPDH (unlabeled primers) as internal endogenous controls.Histology and lectin staining. Hearts were perfused, processed forhistology, and stained with hematoxylin and eosin, trichrome, orTriticum vulgaris lectin TRITC conjugate as previously described(49). Myocyte area was determined using NIH ImageJ (version1.38X) based on photomicrographs of a standard graticle ruler.Western immunoblots. Human ventricular (50 g) samples wereprepared in denaturing sample loading buffer, separated by 8% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. Themembrane was incubated overnight at 4°C in 5% milk and Trisbufferedsaline-Tween with polyclonal goat anti-MuRF1 antibody(NB100-2406, Novus Biologicals, Littleton, CO). The membrane waswashed, incubated with horseradish peroxidase (HRP)-conjugatedanti-goat antibody (sc-2768, Santa Cruz Biotechnology, Santa Cruz,CA), and washed again. The HRP signal was then detected using theECL Plus Western Blotting system (RPN2132, GE Healthcare) accordingto the manufacturer’s protocol.Statistical analysis. One-way ANOVA or Student’s t-test wasperformed using Sigma Stat 3.5 (Systat Software, San Jose, CA) andbasic statistics on Microsoft Excel 2007 (Microsoft, Seattle, WA).Results are expressed as means SE, with statistical significancedefined as P 0.05.RESULTSCardiac MuRF1 increases after cardiac unloading. Theplacement of a LVAD in patients waiting for a heart transplanthelps improve cardiac output by taking over the pumping ofblood, effectively unloading the work the heart has to do. Anumber of studies have reported that LVAD-induced unloadingresults in beneficial cardiac atrophy, which is evidencedby a reduction in LV mass (4, 31, 40, 53). In thisstudy, we collected samples from six patients before andafter the placement of a LVAD, including two patients withmatched consecutive samples (Fig. 1A). Post-LVAD levelsof cardiac MuRF1 protein were significantly elevated(60%) compared with MuRF1 protein levels found insamples taken pre-LVAD (Fig. 1B). In the two matchedsamples, post-LVAD cardiac MuRF1 levels increased 52%and 39% from pre-LVAD levels taken from adjacent tissue(Fig. 1A). This suggested that MuRF1 was intrinsicallyinvolved in the cardiac atrophy resulting from LVADinducedcardiac unloading. This result led us to propose thehypothesis that MuRF1 is involved in the regulation ofcardiac atrophy. To test this hypothesis, we challengedMuRF1 / mice to two independent models of cardiacatrophy: 1) cardiac hypertrophy regression after reversal ofTAC and 2) dexamethasone-induced atrophy.MuRF1 is necessary for the reversal of pathological cardiachypertrophy in vivo. We (49) have recently identified thathearts taken from adult MuRF1 / mice are indistinguishablefrom WT littermate controls. We (49) also discovered thatMuRF1 / mice develop an exaggerated cardiac hypertrophyafter the induction of pressure overload by TAC, suggestingthat MuRF1 antagonizes the development of pathological cardiachypertrophy. In the present study, we investigated the rolethat MuRF1 plays in the cardiac atrophy that is seen with theDownloaded from ajpheart.physiology.org on May 11, 2009AJP-Heart Circ Physiol • VOL 296 • APRIL 2009 • www.ajpheart.org
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